Compositions and methods related to tauopathy

ABSTRACT

Disclosed herein are isolated polypeptides, antibody preparations, treatment methods, diagnostic methods, and screening methods related to tauopathy. Generally, the isolated polypeptide includes a core pentapeptide, with the proviso that the isolated polypeptide is not a native full-length tau protein. Generally, the antibody preparations include antibody that specifically binds to SEQ ID NO:12. Generally, the treatment methods include administering to a subject a composition that includes the isolated polypeptide. Generally, the diagnostic methods includes contacting a sample from a subject with an antibody preparation that includes antibody that specifically binds to SEQ ID NO:12, and then detecting a ligand in the sample that specifically binds the antibody preparation. Generally, the screening method includes incubating a mixture of caspase-2, a labeled caspase-2 cleavage substrate, and a test compound under conditions effective to permit the caspase-2 to cleave the caspase-2 cleavage substrate, then determining whether the test compound inhibits cleavage of the substrate by caspase-2.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/738,510, filed Dec. 18, 2012, which is incorporated hereinby reference.

GOVERNMENT FUNDING

This invention was made with government support under R01-NS063214awarded by the National Institutes of Health. The government has certainrights in the invention.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submitted tothe United States Patent and Trademark Office via EFS-Web as an ASCIItext file entitled “110-03950101_SequenceListing_ST25.txt” having a sizeof 25 kilobytes and created on Dec. 16, 2013. The information containedin the Sequence Listing is incorporated by reference herein.

BACKGROUND

Alzheimer's disease is characterized by the accumulation of amyloidplaques, composed of Aβ proteins, and neurofibrillary tangles, composedof tau proteins. It can be genetically linked to mutations that increasethe propensity for Aβ to form pathogenic aggregates; interestingly,however, it is never linked to mutations in tau. An implication of thisgenetic dichotomy is that the formation of a pathogenic form of Aβ, butnot tau, initiates Alzheimer's disease. Aβ*56 (Abeta star 56) has beenproposed to be the pathogenic form of Aβ that initiates Alzheimer'sdisease.

Aβ*56 requires tau to impair memory function. Tau is therefore an Aβeffector molecule. The clinical observation that amyloid plaques depositprior to abnormal increases in spinal fluid tau is consistent with thisidea. These and other related results indicate that the pathogenesis ofAlzheimer's disease requires both the Aβ and tau proteins.

SUMMARY OF THE INVENTION

In one aspect, this disclosure provides an isolated polypeptide thatincludes a core pentapeptide, with the proviso that the isolatedpolypeptide is not a native full-length tau protein. In someembodiments, the isolated polypeptide of claim 1 further comprising atleast one amino acid appended to the N-terminus of the corepentapeptide. In other embodiments, the isolated polypeptide of eitherclaim 1 or claim 2 further comprising at least one amino acid appendedto the C-terminus of the core pentapeptide. In some embodiments, theisolated polypeptide can be a peptidomimetic of the isolated polypeptideof any preceding embodiment.

In some embodiments, the core pentapeptide can be SEQ ID NO:1.

In another aspect, this disclosure provides a composition that includesthe isolated polypeptide of any preceding embodiments and apharmaceutically acceptable carrier.

In another aspect, this disclosure provides a method that generallyincludes administering to a subject a composition that includes anisolated polypeptide of any embodiment described above. In some cases,the method can involve administering to a subject having or at risk ofhaving a tauopathy an amount of the composition effective to inhibit atleast one of the following: caspase-2-dependent cleavage of tau,production of TCP30, production of TCP35, or production of TCP40. Inother cases, the method can involve administering to a subject having atauopathy an amount of the composition effective to ameliorate at leastone clinical sign or symptom characteristic of a tauopathic condition.In still other cases, the method can involve administering to a subjectat risk of having a tauopathy an amount of the composition effective toprotect the subject against development of a tauopathic condition.

In some embodiments, the tauopathic condition can include Alzheimer'sdisease.

In another aspect, this disclosure provides methods of screeningcompounds. In some embodiments, the method can include providing amixture of caspase-2, a test compound, and a labeled caspase-2 cleavagesubstrate; incubating the mixture under conditions effective forcaspase-2 to cleave the caspase-2 cleavage substrate in the absence ofthe test compound to produce a labeled caspase-2 cleavage product;detecting at least one of: the uncleaved labeled caspase-2 cleavagesubstrate or the labeled caspase-2 cleavage product; and identifying thetest compound as an inhibitor of caspase-2 if at least one of thefollowing is true: the uncleaved labeled caspase-2 cleavage substrate isdetected in an amount greater than a first predetermined referencevalue, or the labeled caspase-2 cleavage product is detected in anamount less than a second predetermined reference value. In otherembodiments, the method can include providing a mixture of a labeledcaspase-2 cleavage substrate, a labeled caspase-2 cleavage product in apredetermined ratio to the caspase-2 cleavage substrate, caspase-2, atest compound; incubating the mixture under conditions effective forcaspase-2 to cleave the caspase-2 cleavage substrate in the absence ofthe test compound to produce the labeled caspase-2 cleavage product;determining the ratio of the caspase-2 cleavage substrate to thecaspase-2 cleavage product; and identifying the test compound as aninhibitor of caspase-2 if the ratio of caspase-2 cleavageproduct:caspase-2 cleavage substrate is less than a reference ratio ofcaspase-2 cleavage product:caspase-2 cleavage substrate in the absenceof the test compound.

In some cases of either embodiment of the screening methods, the labeledcaspase-2 cleavage substrate comprises SEQ ID NO:2 and/or the caspase-2cleavage product comprises SEQ ID NO:3. In other cases of eitherembodiment of the screening methods, the labeled caspase-2 cleavagesubstrate comprises SEQ ID NO:4 and/or the caspase-2 cleavage productcomprises SEQ ID NO:5.

In another aspect, this disclosure provides an antibody preparation thatincludes antibody that specifically binds to SEQ ID NO:10. In someembodiments, the antibody can include polyclonal antibody. In otherembodiments, the antibody can include a monoclonal antibody.

In another aspect, this disclosure provides a method that generallyincludes contacting a biological sample fluid from a subject thatincludes cerebrospinal with an antibody preparation that includesantibody that specifically binds to SEQ ID NO:10 and detecting a ligandin the sample that specifically binds the antibody preparation. In someembodiments, the ligand can include the amino acid sequence of SEQ IDNO:10. In some embodiments, the subject may exhibit at least one symptomor clinical sign of a tauopathic condition. In other embodiments, thesubject need not exhibit at least one symptom or clinical sign of atauopathic condition.

In another aspect, this disclosure provides an aptamer preparation thatincludes one or more aptamers that specifically bind to SEQ ID NO:10.

In yet another aspect, this disclosure provides a method that generallyincludes contacting a biological sample from a subject with an aptamerpreparation that includes one or more aptamers that specifically bind toSEQ ID NO: 10. In some embodiments, the biological sample can includescerebral spinal fluid.

The above summary is not intended to describe each disclosed embodimentor every implementation of the present invention. The description thatfollows more particularly exemplifies illustrative embodiments. Inseveral places throughout the application, guidance is provided throughlists of examples, which examples can be used in various combinations.In each instance, the recited list serves only as a representative groupand should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Inverse correlation between memory function and TCP35 levels.Spatial reference memory of 6-month (M) rTg510 mice in which tauexpression was suppressed by doxycycline until they were 3M was assessedusing the Morris water maze assay. Mice were then killed and TCP35levels in total forebrain homogenates were measured by immunoblotting.(A) Mean platform score and (B) platform crossing index correlatedinversely with TCP35 levels. Mean platform score is the average %-timeof four probe trials. Platform crossing index is the number of targetplatform crossings minus the average number of non-target platformcrossings.

FIG. 2. The expression of TCP35 parallels the development of cognitiveimpairment. The western blot panels (A) show full length tau (55 kDa)and TCP35 in the hippocampus of rTg4510 mice from 1.3 to 4.5 months ofage (probed with antibody Tau-5 recognizing mouse and human tau). PanelsB and C show the quantification of age-dependent TCP35 expression (B,densitometry) and the development of memory impairment (C, percent timein the target quadrant).

FIG. 3. TCP35 is an N-terminal fragment of tau. Representativeimmunoblots of tau proteins in forebrain homogenates of TgNeg andrTg4510 mice. (A) TCP35 can be detected by Tau-13 mAb, whichspecifically recognizes amino acid residues 2-18 in human tau, (B) butnot by T46 mAb, which recognizes an epitope near the last 38 amino acidresidues (404-441 aa) on the C-terminus of tau. βIII-tubulin was probedto ensure loading consistency.

FIG. 4. Identification of caspase-2 cleavage site in tau. Full-length(FL) tau and truncation mutant proteins that are missing the C-terminusafter four aspartate residues mimicking tau cleaved at D283, D295, D314and D348 were synthesized from the corresponding cDNAs using the TNT T7Quick Coupled Transcription/Translation System. Immunoblotting usingTau-13 mAb showed that tau truncation mutant ΔC314 is closest to 35 kDain molecular weight.

FIG. 5. Caspase-2 cleaves tau in vitro, producing a 35 kDa fragment.Recombinant caspases -1, -2, -3, -6, -7, -8, -9, or -10 were incubatedat 37° C. for 2 hours with tau proteins derived from three differentsources. Immunoblots using Tau-13 mAb showed a 35 kDa fragment generatedby co-incubating caspase-2 with full-length tau proteins purified from(A) brain lysates of Tg4510 mice expressing human tau_(P301L), (B) brainlysates of rTg21221 mice expressing human wild-type tau (tau_(wT))(Hoover et al., Neuron, 2010), or (C) synthetic human tau_(WT). 50 μMz-VAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone;Sigma-Aldrich; St. Louis, Mo.) (+inhibitor), a general caspaseinhibitor, blocked all cleavage. Caspases-1 and -8 may cleave tauproteins near the N-terminus and remove Tau-13 mAb epitopes, preventingdetection of proteolytic products. Arrows denote the 35 kDa fragment,and arrowheads denote the tau fragment truncated at D421.

FIG. 6. A 35 kDa fragment is produced by caspase-2-mediated cleavage atD314 in vitro. Incubation of recombinant caspase-2 with wild-type tau ortau mutants, tau_(D314E) and tau_(D314G), that resist caspase cleavage,synthesized from the corresponding cDNAs using the TNT T7 Quick CoupledTranscription/Translation System, generated a 35 kDa fragment only inthe wild-type tau reaction that comigrates with tau truncation mutantΔC314. The arrow denotes the 35 kDa fragment. The arrowhead denotes thetau D421 cleavage product.

FIG. 7. Cleavage-specific polyclonal antibodies H1485 recognize a 35 kDaband in rTg4510 brain. H1485 antibodies were generated against theneo-epitope of caspase-2 cleaved tau at D314. rTg4510 brain lysate wasimmunoprecipitated with Tau-13 mAb. The immunoprecipitates were detectedon immunoblots with H1485 antibodies, revealing a 35 kDa band in rTg4510brain (arrow).

FIG. 8. TauAC314 mislocalizes to dendritic spines. Images of rathippocampal neurons co-expressing DsRed, which permeates all dendriticspines and shafts permitting their visualization, and GFP-taggedfull-length tau (FL) or the tauΔC314 mutant, show aberrant targeting oftauΔC314 to dendritic spines.

FIG. 9. Effects of proline-directed phosphorylation on in vitro TCP35generation. Wild-type (WT), phosphorylation-deficient (AP) andpseudophosphorylated (EP) tau proteins are synthesized from thecorresponding cDNAs in vitro by using the TNT T7 Quick CoupledTranscription/Translation System. Recombinant caspase-2 was incubatedwith tau proteins to generate TCP35 and the reactions were stopped atthe indicated time points. TCP35 was immunoblotted with Tau-13 mAb.

FIG. 10. Caspase-2-mediated cleavage of Asp314 peptide substrate.Synthetic peptide substrate FITC-AHX-GSVQIVYKPVDLSKVTS-COOH was designedbased on amino acids 304-320 of tau protein that encompasses Asp314cleavage site. The peptide substrate was cleaved by coincubating withrecombinant caspase-2 and was separated from cleavage product by usingLabChip drug discovery system. The fluorescence emitted by FITC groupwas monitored to measure peptide quantity.

FIG. 11. Caspase inhibitor blocks caspase-2-mediated cleavage of Asp314peptide substrate. Synthetic peptide substrate was coincubated withrecombinant caspase-2 in the presence of general caspase inhibitorz-VAD-fmk. The fluorescence of the substrate peptide was measured byusing LabChip drug discovery system.

FIG. 12. TCP35 is present in human cerebrospinal fluid. Tau proteinswere immunoprecipitated from human cerebrospinal fluid with Tau-13 mAb.The immunoprecipitates were detected on immunoblots with Tau-13 andH1485 antibodies, respectively, revealing TCP35 bands (arrow) in samples#266 and #267A. Tau truncation mutant ΔC314 was probed to show thecomigration with TCP35.

FIG. 13. Profile of tau cleavage product in human brain lysates. Humanbrain lysates were probed with cleavage site-specific antibody H1485,3-repeat isoform antibody 8E6/C11, 4-repeat isoform antibody 1E1/A6 andtotal tau antibody Tau-13. Three tau fragments TCP40, TCP35 and TCP30were recognized by H1485 antibody.

FIG. 14. Tau cleavage product levels increase in patients with mildcognitive impairment and Alzheimer's disease. The protein levels of (A)TCP35, (B) TCP40 and (C) TCP30 were quantified, respectively. (D) Thetotal protein levels of TCP35, TCP40 and TCP30 (TCP) were alsoquantified.

FIG. 15. Images of rat hippocampal neurons coexpressing Dsred andGFP-tagged wild-type tau or tau P301L mutant. Neurons were treated withcaspase-2 inhibitor Ac-VDVAD-CHO or caspase-3 inhibitor Ac-DEVD-CHO for1 day.

FIG. 16. Quantification of total spines and GFP-tau-containing spines inneurons coexpressing DsRed and GFP-tau.

FIG. 17. Excitatory Synaptic Transmission in rTg4510 neurons treatedwith caspase-2 inhibitor. (A) Mean mEPSC amplitudes of rTg4510 neurons(−/− and +/+) treated with 20 μM caspase-2 inhibitor (Ac-VDVAD-CHO; SEQID NO:35) for two hours. Caspase-2 inhibitor significantly increased themean amplitude of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid) mEPSCs in rTg4510 (+/+) neurons compared to vehicle (Veh) control.Caspase-2 inhibitor had no effect on rTg4510 (−/−) neurons. (B) MeanmEPSC frequency of rTg4510 neurons (−/− and +/+) treated with 20 μMcaspase-2 inhibitor for two hours. Caspase-2 inhibitor had no effect onrTg4510 neurons (−/− or +/+) frequencies.

FIG. 18. (A) Design of pentapeptide inhibitors of caspase-2 (cleavagesequence SEQ ID NO:1 and canonical sequence SEQ ID NO:35). (B)Generalized structure of pentapeptide inhibitors of caspase-2, wherewith R₁=Ac, aa_(x)=side chains of naturally-occurring D-amino-acids orL-amino-acids, and R₂=a group that covalently links to the caspaseactive site cysteine. (C) Exemplary chemical groups that covalently linkto the caspase active site cysteine.

FIG. 19. (A) Generalized structure of peptidomimetics related to theoriginally-designed pentapeptide inhibitor of caspase-2, whereR₁=acetyl, acyl, aryl, heteroaryl, diaryl, biaryl, aralkyl, variouslysubstituted; aa_(x)=D- or L-amino-acids, or unnatural amino acids. aa₁can be replaced as reflected in (B), where R₅ is COOH, COR, CO₂R, CN,OCO₂R, NO₂, NCOR, SCOR, SOR, SO₂R, NHSO₂R, NHCOR, or tetrazole; X ispeptidic carbamoyl group, i.e., —CONH—, can be replaced bypeptidomimetic bonds such as —CH₂NH—, —COCH₂, —CSNH—, and retro-inversobonds or oxazole, imidazole and thiophene, variously substituted; and R₂is as shown in (C) or can be acetyl, acyl, aryl, heteroaryl, diaryl,biaryl, aralkyl variously substituted.

FIG. 20. Generalized design of macrocyclic/stapled peptides based on i,i+3, i+4, and i+7 strategies, where L=Linker (e.g., linkers described inInternational Publication No. WO 2013/123266 A1), and X is O or S.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Although a great deal is known about the neuropathology of tauopathy,much less is understood about the molecular processes that cause theclinical symptoms. Consequently, nearly all research to develop newtreatments for Alzheimer's disease has been directed at ameliorating theeffects of neuronal loss and reducing neuropathology. This has led tothe development of medications that temporarily improve symptoms and/orclinical signs of the disease but do not address the underlying cause ofthe disease.

Much research on tau still aims to understand how it kills neurons andhow it aggregates to form neurofibrillary tangles. In contrast, the workdescribed herein addresses the deterioration in brain function thattakes place before neuron loss occurs and is unrelated toneurofibrillary tangles, by specifically addressing the basic questionof the form of tau (here called tau*) initiates memory loss intauopathy.

As noted in the background, the pathogenesis of Alzheimer's diseaserequires both the Aβ and tau proteins. We have determined that specificforms of Aβ and tau proteins are involved. For example, Aβ*56 caninitiate the disease long before the degenerative processes progress tothe point of diagnostic certainty, and tau* (tau star) causes theclinical symptoms of Alzheimer's disease that are not due to neuronalloss. We have, therefore, investigated tau*, the molecular species oftau involved in initiating the clinical symptoms of tauopathy. We reportidentifying tau* and a potential molecular mechanism leading to itsformation.

We identify markers that have not been previously implicated in thepathogenesis of Alzheimer's disease or other tauopathy. The markers aregenerated when caspase-2 cleaves various isoforms of tau at aspartate314 (D314). One marker, TCP35, has an approximate molecular weight of 35kDa. TCP30 has an apparent molecular weight of 30 kDa. TCP40 has anapparent molecular weight of 40 kDa. Caspase-2, while involved inAP-related neurotoxicity, has not been associated with tau-relatedneural dysfunction. Moreover, we have identified the cleavage site atwhich caspase-2 cleaves tau, designed an inhibitor ofcaspase-2-dependent tau cleavage that may have therapeutic potential,and designed high throughput assays for the identification of additionalinhibitors of caspase-2-dependnet tau cleavage.

Thus, in one aspect, this disclosure provides a composition thatincludes an isolated polypeptide that comprises an amino acid sequencedesigned to interfere with caspase-2-dependent tau cleavage. As usedherein, “isolated” and variations thereof refer to a polypeptide thathas been removed from its natural environment to any degree. Forinstance, an isolated polypeptide is a polypeptide that has been removedfrom the cytoplasm and/or any membrane of a cell, and many of thepolypeptides, nucleic acids, and other cellular material of its naturalenvironment are no longer present. As such chemically synthesizedpolypeptides are, by definitions, removed from their natural environmentand, therefore, “isolated.” The term “isolated” does not convey anyspecific degree to which the other cellular components are removed. Asused herein, “polypeptide” refers to a sequence of amino acid residueswithout regard to the length of the sequence, regardless of whether eachamino acid residue in the sequence is bound to its neighbor through apeptide bond. Therefore, the term “polypeptide” refers to any amino acidsequence having at least two amino acids and includes peptidomimetics.

The initial design of inhibitors of caspase-2 dependent tau cleavageinvolved sequential changes of the amino acids in the cleavage sequenceand leading to the canonical cleavage sequence reported in theliterature. This allows the generation of novel linear, reversible,covalent, pentapeptide inhibitors. In some embodiments, however, thepentapeptide inhibitors may be designed to include a group thatinteracts with, for example, the cysteine residue at the caspase-2active site in a noncovalent manner. The “canonical cleavage sequence”is the sequence of five amino acids (VDVAD, SEQ ID NO:35) that have beenidentified to constitute the most potent inhibitor of caspase-2, whenthe pentapeptide has the formula VDVAD-X, where X is a chemical groupthat covalently links to the caspase active site cysteine (FIG. 18B). Insome embodiments, X can include, for example, any one of the groupsillustrated in FIG. 18C.

Thus, in some embodiments, the isolated polypeptide can be anypolypeptide that possesses the structure illustrated in FIG. 18.

A designed inhibitor can include one or more further modifications thatcan provide metabolic stability and/or cell permeability.

Table 1 lists exemplary pentapeptides that have been designed andsynthesized based on the general structure:

In competitive binding assays against caspase-2, caspase-3, caspase-6,and caspase-7 (PROMEGA-GLO 2; Promega, Corp., Madison, Wis.), the parentpentapeptide based on the sequence Ac-YKPVD-CHO (SEQ ID NO: 1) exhibiteda K_(i) value of 200 nM (caspase-2), 19.8 μM (caspase-3), 184 μM(caspase-6) and greater than 100 μM (caspase-7). The alternativepentapeptides reflected in Table 1 exhibit similar activity.

TABLE 1 SEQ ID MW R₁ aa₅ aa₄ aa₃ aa₂ aa₁ R₂ NO (Da) cLogP CH₃ Y K P V DCHO 1 646.7 −2.51 CH₃ Y K(Ac) P V D CHO 19 688.8 −2.58 CH₃ Y D P V D CHO20 633.7 −3.03 CH₃ Y S P V D CHO 21 605.6 −3.10 CH₃ Y C P V D CHO 22621.7 −2.28 CH₃ F K P V D CHO 23 630.7 −2.12 CH₃ 4F-F K P V D CHO 24648.7 −1.96 benzyl Y K P V D CHO 25 722.8 −1.47 benzyl Y D P V D CHO 26709.7 −1.75 CH₃ Y K P F D CHO 27 694.8 −1.72 CH₃ Y D P F D CHO 28 681.7−2.24 CH₃ Y K P A D CHO 29 618.7 −3.39 CH₃ Y D P A D CHO 30 605.6 −3.91CH₃ Y K V V D CHO 31 648.8 −1.84 CH₃ Y D V V D CHO 32 635.7 −2.36 CH₃ YK P Y D CHO 33 710.8 −2.11 CH₃ Y K P V D CHO 34 697.7 −2.63 4F-F:4-fluorophenylalanine

Thus, in one particular embodiment, the isolated polypeptide can includethe amino acids Tyr-Lys-Pro-Val-Asp (SEQ ID NO:1). In other particularembodiments, the isolated polypeptide can include the amino acids of anyone or more of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27,SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,SEQ ID NO:33, or SEQ ID NO:34.

The amino acid sequences of the pentapeptides reflected in Table 1 (SEQID NO:1, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO: 24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, and SEQ ID NO:34) were designed to mimic, at least in part, thesite at which caspase-2 cleaves tau. In so doing, the polypeptides caninhibit caspase-2-dependent cleavage of tau and the resulting productionof TCP30, TCP35, and/or TCP40. As used herein, “inhibit” and variationsthereof refer to any measurable reduction of caspase-2-dependentcleavage of tau and/or any measurable reduction in the presence ofTCP30, TCP35, and/or TCP40 (collectively herein for brevity, “caspase-2tau cleavage”). The extent of inhibition may be characterized as apercentage of a normal level of activity. Without wishing to be bound byany particular theory, the isolated polypeptide can occupy the activesite of caspase-2. In some cases, the polypeptide may serve as acompetitive inhibitor of caspase-2 tau cleavage.

In some embodiments, the isolated polypeptide may be variant of apentapeptide reflected in Table 1 that includes one or more additionalamino acids appended to either terminus of core pentapeptide (e.g., SEQID NO:1, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO: 24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, or SEQ ID NO:34) so long as the variant polypeptide retains theability to inhibit caspase-2 tau cleavage. Molecular modeling algorithmsmake it routine for one to determine whether any particular variant of acore pentapeptide will adopt a conformation that will allow the variantto inhibit caspase-2 tau cleavage. Thus, the isolated polypeptide caninclude an addition of, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues appended toeither terminus of a core pentapeptide. In certain embodiments, theisolated polypeptide can include an addition of, for example, no morethan 10 amino acid residues to either terminus of a core pentapeptide.In embodiments in which the isolated polypeptide includes an addition toeach terminus of a core pentapeptide, the length and/or the particularamino acid sequence added to one terminus may be independent of thelength and/or particular amino acid sequence added to the otherterminus.

In some embodiments, the isolated polypeptide also may be a variant of acore pentapeptide that includes one or more conservative substitutionsso long as the variant polypeptide retains the ability to inhibitcaspase-2 tau cleavage. A conservative substitution for an amino acid inthe isolated polypeptide may be selected from other members of the classto which the amino acid belongs. For example, it is well-known in theart of protein biochemistry that an amino acid belonging to a groupingof amino acids having a particular size or characteristic (such ascharge, hydrophobicity and hydrophilicity) can be substituted foranother amino acid without altering the activity of a protein. Forexample, nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine.Polar neutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine and glutamine. The positively charged (basic) aminoacids include arginine, lysine and histidine. The negatively charged(acidic) amino acids include aspartic acid and glutamic acid.Conservative substitutions include, for example, Lys for Arg and viceversa to maintain a positive charge; Glu for Asp and vice versa tomaintain a negative charge; Ser for Thr so that a free —OH ismaintained; and Gln for Asn to maintain a free —NH₂. In someembodiments, the isolated polypeptide also can be designed to provideadditional sequences, such as, for example, the addition of codingsequences for added C-terminal or N-terminal amino acids that mayfacilitate purification by trapping on columns or use of antibodies.Such tags include, for example, histidine-rich tags that allowpurification of polypeptides on nickel columns. Such gene modificationtechniques and suitable additional sequences are well known in themolecular biology arts.

In some embodiments, the isolated polypeptide can include one or morepost-expression biological or chemical modifications of the polypeptidesuch as for example, a glycosylation, an acetylation, a phosphorylation,and the like, or any combination of two or more such modifications.

In some embodiments, the isolated polypeptide can be modified to includea chemical group that can covalently or non-covalently bind to thecysteine residue at the caspase-2 active site. For example, the isolatedpolypeptide can include certain amino acid derivatives that can induceinhibitory interactions with caspases. For example, a core pentapeptidereflected in Table 1, or the polypeptides with conservative amino acidsubstitutions as describe above, can be modified to be peptide-Asp*,where Asp* can include a terminal modification reflected in, forexample, the modifications shown as R₂ in FIG. 18C and 19C. R₂ can be asshown in FIG. 18C or FIG. 19C or can be an acetyl group, an acyl group,an aryl group, a heteroaryl group, a diaryl group, a biaryl group, or anaralkyl group, any of which may be variously substituted In the contextof R₂, “variously substituted” means that there may be one or moresubstituent group present on that specified moiety. Exemplarysubstituent groups include, for example, fluorine (F), chlorine (Cl),bromine (Br), trifluoromethyl (CF₃), alkyl, hydroxy (OH), alkoxy (OR),alkylthio (SR), cyano, carboxyl ester (CO₂R), amino (NH₂) or amido(NHCOR).

In some embodiments, the isolated polypeptide may be modified to includea chemical group reflected as R₁ in FIG. 18B or FIG. 19A. R₁ can bemodified to include an acetyl group, an acyl group, an aryl group, aheteroaryl group, a diaryl group, a biaryl group, or an aralkyl group,any of which may be variously substituted. In the context of R₁,“variously substituted” means that there may be one or more substituentgroup present on that specified moiety such as fluorine (F), chlorine(Cl), bromine (Br), trifluoromethyl (CF₃), alkyl, hydroxy (OH), alkoxy(OR), alkylthio (SR), cyano, carboxyl ester (CO₂R), amino (NH₂) or amido(NHCOR).

Amino acids aa_(i)-aa₅ can be any D-amino acid, L-amino-acid, orunnatural amino acid such as, for example, a pseudo-peptide, adipepsipeptide, or a β3-aza aminoacid. More generally, an unnaturalamino acids can also describe a bioisoteric replacement of the lateralchain of natural amino acids such as, for example, pyridinyl-,naphthyl-, homophenyl-, thienyl-, quinolyl-, pyrimidyl-alanine forphenylalanine; tyrosine or tryptophan amino acid instead of tyrosine;nor-leucine, cycloalkyl-alanine, cylohexyl glycine, spirocyloalkylglycine for leucine, isoleucine, valine, alanine; piperazinyl-alaninefor lysine; thiaproline, 3- and 4-fluoro-, alkoxy-, aryloxy, alkylthio-,amino-proline for proline.

In some embodiments, aa_(i) can be replaced as reflected in FIG. 19B,where R₅ is COOH, COR, CO₂R, CN, OCO₂R, NO₂, NCOR, SCOR, SOR, SO₂R,NHSO₂R, NHCOR, or tetrazole; X is peptidic carbamoyl group, i.e.,—CONH—, can be replaced by peptidomimetic bonds such as, for example,-CH₂NH-, -COCH₂, -CSNH-, and retro-inverso bonds or oxazole, imidazoleand thiophene, any of which can be variously substituted. Exemplarysubstituents can include, for example, fluorine (F), chlorine (Cl),bromine (Br), trifluoromethyl (CF₃), alkyl, hydroxy (OH), alkoxy (OR),alkylthio (SR), cyano, carboxyl ester (CO₂R), amino (NH₂) or amido(NHCOR)).

Additionally, some embodiments of the isolated polypeptide can includeany combination of two or more features of the various embodimentsdescribed above. Moreover, the site of any of the modificationsdescribed above may be to one or more of the amino acid residues of acore pentapeptide or, in some cases, to amino acids residues that arepart of an addition to either the C-terminus or the N-terminus of a corepentapeptide.

In some embodiments, the isolated polypeptide can include a prodrugmodification. As used herein, a “prodrug modification” refers to aderivative of an isolated polypeptide as described herein that canundergo a chemical or enzymatic biotransformation, thereby releasing theactive isolated polypeptide in the body. Various conventional prodrugmodifications are known in the pharmaceutical arts.

In some embodiments, the isolated polypeptide may be a peptidomimetic.As used herein, the term “peptidomimetic” refers to a polypeptidedesigned to mimic a template polypeptide, but includes at least onenon-naturally occurring modification that results is in conferring afavorable characteristic to the peptidomimetic. Various conventionalmodifications of this sort—e.g., such as linking of amino acid sidechains, substitution of atoms in the peptide backbone, the replacementof amino acids with aromatic rings or heteroaromatic rings, substitutinga D-amino acid residue for an L-amino acids residues, or substituting anon-peptide linkage for a peptide linkage between adjacent amino acidresidues—are known in the pharmaceutical arts. Exemplary non-peptidelinkages include, for example, —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH—(cisand trans), —COCH₂—, —CH(OH)CH₂—, or —CH₂SO—. A peptidomimetic maypossess one or more advantages over a natural polypeptide including, forexample, more economical production, greater chemical stability, alteredspecificity, or enhanced pharmacological properties such as half-life,absorption, potency, or efficacy.

In another aspect, this disclosure provides compositions that includethe isolated polypeptide described above. Any embodiment of the isolatedpolypeptide may be a component of such a composition.

The composition described herein can include a “pharmaceuticallyacceptable carrier.” As used herein, “carrier” includes any solvent,dispersion medium, vehicle, coating, diluent, antibacterial, and/orantifungal agent, isotonic agent, absorption delaying agent, buffer,carrier solution, suspension, colloid, and the like. The use of suchmedia and/or agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the isolated polypeptide, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions. As used herein, “pharmaceuticallyacceptable” refers to a material that is not biologically or otherwiseundesirable, i.e., the material may be administered to an individualalong with the isolated polypeptide without causing any undesirablebiological effects or interacting in a deleterious manner with any ofthe other components of the pharmaceutical composition in which it iscontained.

A pharmaceutical composition that includes the isolated polypeptide maybe prepared as any suitable pharmaceutical formulation. Thepharmaceutical formulation may be any suitable form adapted to apreferred route of administration. Thus, a composition can be formulatedto be administered via known routes including, for example, oral,parenteral (e.g., intradermal, transcutaneous, subcutaneous,intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g.,intranasal, intrapulmonary, intramammary, intravaginal, intrauterine,intradermal, transcutaneous, rectally, etc.). It is foreseen that acomposition can be administered to a mucosal surface, such as byadministration to, for example, the nasal or respiratory mucosa (e.g.,by spray or aerosol). Also, a composition also can be administered via asustained or delayed release.

A formulation may be conveniently presented in unit dosage form and maybe prepared by methods well known in the art of pharmacy. Methods ofpreparing a composition with a pharmaceutically acceptable carrierinclude the step of bringing the isolated polypeptide into associationwith a carrier that constitutes one or more accessory ingredients. Ingeneral, a formulation may be prepared by uniformly and/or intimatelybringing the active compound into association with a liquid carrier, afinely divided solid carrier, or both, and then, if necessary, shapingthe product into the desired formulations.

The isolated polypeptide may be provided in any suitable form includingbut not limited to a solution, a suspension, an emulsion, a spray, anaerosol, or any form of mixture. The composition may be delivered informulation with any pharmaceutically acceptable excipient, carrier, orvehicle. For example, the formulation may be delivered in a conventionaltopical dosage form such as, for example, a cream, an ointment, anaerosol formulation, a non-aerosol spray, a gel, a lotion, and the like.The formulation may further include one or more additives including suchas, for example, an adjuvant, a skin penetration enhancer, a colorant, afragrance, a flavoring, a moisturizer, a thickener, and the like.

Thus, in another aspect, this disclosure provides methods of providingtreatment for tauopathy. As used herein, “treat,” “treatment,” orvariations thereof refer to reducing, ameliorating, or resolving, to anyextent, at least one symptom or clinical sign related to a tauopathiccondition. As used herein, “ameliorate” refers to any reduction in theextent, severity, frequency, and/or likelihood of a symptom or clinicalsign characteristic of a particular condition. The treatments describedherein may be prophylactic and/or therapeutic. As used herein,“prophylactic” and variations thereof refer to a treatment that limits,to any extent, the development and/or appearance of a symptom orclinical sign of a tauopathic condition. As used herein, “therapeutic”and variations thereof refer to a treatment that ameliorates one or moreexisting symptoms or clinical signs associated with a tauopathiccondition. As used herein, “sign” or “clinical sign” refers to anobjective physical finding relating to a tauopathic condition capable ofbeing found by one other than the patient. As used herein, “symptom”refers to any subjective evidence of tauopathic condition. The “subject”receiving treatment according to the methods described herein caninclude, for example, animals such as, but not limited to, humans,non-human primates, rodents, dogs, cats, horses, pigs, sheep, goats, orcows.

In some embodiments, the method generally includes administering to asubject having or at risk of having a tauopathic condition an amount ofa pharmaceutical composition as described herein effective to inhibit toany of degree caspase-2 tau cleavage. In other embodiments, the methodgenerally includes administering to a subject having or at risk ofhaving a tauopathic condition an amount of a pharmaceutical compositionas described herein effective to ameliorate at least one clinical signor symptom characteristic of the tauopathic condition. In otherembodiments, the method generally includes administering to a subjecthaving or at risk of having a tauopathic condition an amount of apharmaceutical composition as described herein effective to protect thesubject against development of a tauopathic condition.

As used herein, “at risk” refers to a subject that may or may notactually possess the described risk. Thus, for example, a subject “atrisk” for developing a tauopathic condition is a subject that possessesone or more indicia of increased risk of having, or developing, thespecified condition compared to individuals who lack the one or moreindicia, regardless of the whether the subject manifests any symptom orclinical sign of having or developing the condition. Exemplary indiciaof tauopathic conditions can include, for example, mutations in certaingenes (e.g., APP, PSEN1, PSEN2, CHMP2B, FUS, GRN, MAPT, TARDBP, VCP,and/or the APOE4 variant of APOE) and/or a family history of Alzheimer'sdisease or frontotemporal dementia. As used herein, “protect” refers toany delay in the onset of at least one symptom or clinical signcharacteristic of a particular condition, or any reduction in theextent, severity, frequency, and/or likelihood of the onset of at leastone symptom or clinical sign characteristic of a particular condition.

The amount of the isolated polypeptide administered can vary dependingon various factors including, but not limited to, the specific isolatedpolypeptide, the weight, physical condition, and/or age of the subject,and/or the route of administration. Thus, the absolute weight ofisolated polypeptide included in a given unit dosage form can varywidely, and depends upon factors such as the species, age, weight andphysical condition of the subject, as well as the method ofadministration. Accordingly, it is not practical to set forth generallythe amount that constitutes an amount of the isolated polypeptideeffective for all possible applications. Those of ordinary skill in theart, however, can readily determine the appropriate amount with dueconsideration of such factors.

In some embodiments, the method can include administering sufficient[active agent] to provide a dose of, for example, from about 100 ng/kgto about 50 mg/kg to the subject, although in some embodiments themethods may be performed by administering the isolated polypeptide in adose outside this range. In some of these embodiments, the methodincludes administering sufficient isolated polypeptide to provide a doseof from about 10 μg/kg to about 5 mg/kg to the subject, for example, adose of from about 100 μg/kg to about 1 mg/kg.

Alternatively, the dose may be calculated using actual body weightobtained just prior to the beginning of a treatment course. For thedosages calculated in this way, body surface area (m²) is calculatedprior to the beginning of the treatment course using the Dubois method:m²=(wt kg^(0.425)×height cm^(0.725))×0.007184.

In some embodiments, the method can include administering sufficientisolated polypeptide to provide a dose of, for example, from about 0.01mg/m² to about 10 mg/m².

In some embodiments, the isolated polypeptide may be administered, forexample, from a single dose to multiple doses per week, although in someembodiments the method can be performed by administering the isolatedpolypeptide at a frequency outside this range. In certain embodiments,the isolated polypeptide may be administered from about once per monthto about five times per week.

In another aspect, we provide herein a novel assay for identifyingadditional inhibitors of caspase-2 tau cleavage. We have synthesizednovel fluorescently N-terminal tagged polypeptides and used these noveltagged polypeptides to develop an assay that identifies inhibitors ofcaspase-2 tau cleavage. The assay involves combining a substratepolypeptide (e.g., SEQ ID NO:2 or SEQ ID NO:4) with caspase-2 and a testcompound under conditions effective to allow the caspase-2 to cleave thesubstrate in the absence of the test compound. After a suitableincubation time, one can measure the mobility shift of the cleaved(product) and un-cleaved (substrate) peptides (e.g., using a CaliperLC3000 instrument). This assay is suitable for a high throughputscreening campaign to identify novel compounds that inhibitcaspase-2-dependent cleavage of the substrate.

Exemplary substrate/product polypeptide pairs used in the assay includeFITC-AHX-GSVQIVYKPVDLSKVTS-COOH (SEQ ID NO:2) andFITC-AHX-GSVQIVYKPVD-COOH (SEQ ID NO:3), in which SEQ ID NO:3 is thecaspase-2 cleavage product of substrate SEQ ID NO:2. In otherembodiments, the substrate/product polypeptide pair can includeFITC-AHX-GSVQIVYK(acetyl)PVDLSKVTS-COOH (SEQ ID NO:4) andFITC-AHX-GSVQIVYK(acetyl)PVD-COOH (SEQ ID NO:5), in which SEQ ID NO:5 isthe caspase-2 cleavage product of substrate SEQ ID NO:4. The exemplarysubstrate/product polypeptide pairs are shown with FITC as a fluorescentlabel. However, the assay may be performed using any other suitablelabel or tag conventional for high throughput screening methods.

In some embodiments, the one or both polypeptides of the exemplarysubstrate/product polypeptide pairs identified immediately above may bea variant of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 thatincludes one or more additional amino acids appended to either terminusof the indicated amino acid sequence so long as the substrate/productpair retains the stated caspase-2 cleavage substrate/productrelationship. Molecular modeling algorithms make it routine for one todetermine whether any particular variants of a substrate/product pairwill adopt a conformation that will allow the variants to retain acaspase-2 cleavage substrate/product relationship. Thus, each member ofa substrate/product pair can include an addition of, for example, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aminoacid residues appended to either terminus of SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, or SEQ ID NO:5, with the proviso that the appended aminoacid residues do not introduce additional caspase-2 cleavage sites.

In some embodiments, one or both polypeptides of a substrate/productpair may be a variant of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ IDNO:5 that includes one or more conservative substitutions so long as thesubstrate/product pair retains the stated caspase-2 cleavagesubstrate/product relationship. A conservative substitution for an aminoacid in a substrate polypeptide or product polypeptide may be selectedfrom other members of the class to which the amino acid belongs. Forexample, it is well-known in the art of protein biochemistry that anamino acid belonging to a grouping of amino acids having a particularsize or characteristic (such as charge, hydrophobicity andhydrophilicity) can be substituted for another amino acid withoutaltering the activity of a protein. For example, nonpolar (hydrophobic)amino acids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and tyrosine. Polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Conservative substitutionsinclude, for example, Lys for Arg and vice versa to maintain a positivecharge; Glu for Asp and vice versa to maintain a negative charge; Serfor Thr so that a free —OH is maintained; and Gln for Asn to maintain afree —NH₂.

In some embodiments, the one or both polypeptides of a substrate/productpair may be designed to provide additional sequences, such as, forexample, the addition of coding sequences for added C-terminal orN-terminal amino acids that would facilitate purification by trapping oncolumns or use of antibodies. Such tags include, for example,histidine-rich tags that allow purification of polypeptides on nickelcolumns. Such gene modification techniques and suitable additionalsequences are well known in the molecular biology arts.

In some embodiments, the one or both polypeptides of a substrate/productpair can include one or more post-expression modifications of thepolypeptide such as for example, a glycosylation, an acetylation, aphosphorylation, and the like, or any combination of two or more suchmodifications. Here again, such modifications are permissible so long asthe substrate/product pair retains the stated caspase-2 cleavagesubstrate/product relationship.

Additionally, in some embodiments, one or both of the polypeptides in asubstrate/product pair can include any combination of two or morefeatures of the various embodiments described above. Within asubstrate/product pair, however, it is not necessary that the substratepolypeptide and the product polypeptide include identical modificationsso long as the variant polypeptides retain their caspase-2 cleavagesubstrate/product relationship.

Since acetylation is a known mechanism of protein activity modulation,substrate SEQ ID NO:4 (or variants of SEQ ID NO:4 as describedimmediately above), for example, may be used in a similar mobility shiftassay to detect deacetylation inhibitors. Alternatively, inhibitors of atransacetylase could be detected using the mobility shift of theappropriate paired sets of fluorescently tagged peptides anddeacetylases and transacetylases.

In another aspect, this disclosure provides antibody preparations thatinclude antibody that specifically binds to SEQ ID NO: 10 such as, forexample, SEQ ID NO:12. As used herein, the term “antibody” without anarticle or adjectival modifier, generically refers to preparations thatcan include either a homogenous population of a single immunoglobulinmolecule (e.g., a monoclonal antibody preparation) or a heterogeneouspopulation of a plurality of immunoglobulin molecules (e.g., apolyclonal antibody preparation). As used herein, “specifically binds”and variations thereof refer to the character of exhibiting differentialor a non-general (i.e., non-specific) affinity, to any degree, for aparticular target such as, for example, SEQ ID NO:12. Thus, the term“specifically binds” does not imply or suggest that universallyexclusive specific binding is required in order for, for example, anantibody to “specifically binds” a particular target.

Thus, the antibody preparation may be a polyclonal antibody preparationthat specifically binds to SEQ ID NO:12 such as, for example, H1485,described in detail in the Examples section, below. Alternatively, theantibody preparation can include a monoclonal antibody that specificallybinds to SEQ ID NO:12. The production of a monoclonal antibody thatspecifically binds to a particular polypeptide target is routine forthose of skill in the art once the polypeptide target is identifiedusing conventional methods.

In yet another aspect, this disclosure provides a method that generallyincludes contacting a biological sample from a subject that includescerebral spinal fluid with an antibody preparation that includesantibody that specifically binds to SEQ ID NO:10 or specifically bindsto SEQ ID NO:12, then detecting a ligand in the sample that specificallybinds the antibody preparation. Detecting a ligand in the sample thatspecifically binds the antibody preparation can indicate that thesubject from the sample is obtained has or is at risk of having atauopathic condition. Thus, in some cases, the ligand to which theantibody preparation specifically binds can include the amino acids ofSEQ ID NO:10 or the amino acids of SEQ ID NO:12 including, for example,a product of caspase-2 tau cleavage.

The method may be practiced using a sample obtained from a subject thatexhibits one or more symptoms or clinical signs of a tauopathicconditions. In such circumstances, the method may confirm a diagnosisthat the subject has a tauopathic condition. In other cases, the methodmay be practiced using a sample obtained from a subject that does notexhibit one or more symptoms or clinical signs of a tauopathiccondition. In such circumstances, the method may be used to identify thesubject from whom the sample is obtained as having or at risk of havinga tauopathic condition.

In another aspect, this disclosure provides an aptamer preparation thatincludes one or more aptamers that specifically bind to SEQ ID NO:10such as, for example, SEQ ID NO:12. As used herein, the term “aptamer”without an article or adjectival modifier, generically refers to apreparation that can include either a homogenous population of a singlenucleotide sequence (e.g., a DNA or RNA aptamer preparation) or ahomogenous population of a chemically-modified nucleotide sequence(e.g., a chemically-modified nucleotide aptamer preparation). In thiscontext, too, “specifically binds” and variations thereof refer to thecharacter of exhibiting differential or a non-general (i.e.,non-specific) affinity, to any degree, for a particular target such as,for example, SEQ ID NO: 12. Thus, the term “specifically binds,” in thecontext of an aptamer preparation, does not imply or suggest thatuniversally exclusive specific binding is required in order for, forexample, an aptamer to “specifically bind” a particular target.

Thus, the aptamer preparation may be a nucleotide sequence preparationthat specifically binds to SEQ ID NO:10 or SEQ ID NO:12. Alternatively,the aptamer preparation can include a chemically-modified aptamer basedon a developed nucleotide sequence that specifically binds to SEQ IDNO:10 or SEQ ID NO: 12. The production of a chemically-modified aptamerthat specifically binds to a particular polypeptide target is routinefor those of skill in the art once the parent nucleotide sequence isidentified using conventional methods.

In yet another aspect, this disclosure provides a method that generallyincludes contacting a biological sample from a subject that includescerebral spinal fluid with an aptamer preparation that includes aptamerthat specifically binds to SEQ ID NO:10 or specifically binds to SEQ IDNO:12, then detecting a ligand in the sample that specifically binds theaptamer preparation. Detecting a ligand in the sample that specificallybinds the aptamer preparation can indicate that the subject from thesample obtained, has, or is at risk of having a tauopathic condition.Thus, in some cases, the ligand to which the aptamer preparationspecifically binds can include the amino acids of SEQ ID NO:10 or SEQ IDNO: 12 including, for example, a product of caspase-2 tau cleavage.

The method may be practiced using a sample obtained from a subject thatexhibits one or more symptoms or clinical signs of a tauopathicconditions. In such circumstances, the method may confirm a diagnosisthat the subject has a tauopathic condition. In other cases, the methodmay be practiced using a sample obtained from a subject that does notexhibit one or more symptoms or clinical signs of a tauopathiccondition. In such circumstances, the method may be used to identify thesubject from whom the sample is obtained as having, or at risk ofhaving, a tauopathic condition.

This disclosure further provides a transgenic mouse model (rTg4510,described in the Examples section below) created to study the etiologyof dementia in tauopathy. We found no relationship betweenneurofibrillary tangles and memory loss in rTg4510 mice, suggestingneurofibrillary tangles may represent an attempted neuroprotectivesequestration of tau rather than a causative lesion underlyingimpairment. In addition, the data supported the possibility that the tauspecies responsible for inducing cognitive deficits is a functionalprotein that lies upstream of neurofibrillary tangle formation. The dataindicated that memory function in rTg4510 mice improved when theproduction of soluble forms of tau was inhibited. Therefore, aftereliminating tangles as a cause of memory loss in rTg4510 mice, wefocused our studies on soluble forms of tau that precede the formationof neurofibrillary tangles.

Specifically, we sought to identify soluble forms of tau whoseexpression precedes the formation of tangles. Initially, we identifiedcandidate tau* molecules (normalized densitometry) that inverselycorrelated with retention of spatial reference memory in a water maze,prior to neuron loss or tangle formation. We successfully used a similarstrategy to identify the specific Aβ assembly responsible for thedevelopment of cognitive impairment in Tg2576 mice. We employed a 3-stepprocedure, modified from the one we used to examine Aβ oligomers, toseparate tau peptides into distinct soluble extra/intracellular (S1),membrane-associated (S2) and insoluble pools.

Immunohistochemical (IHC) studies of rTg4510 show a consistent hierarchyfor the expression of pathological tau epitopes, with CP-13 and MC-1representing the earliest biochemical changes in tau. Following the IHCstudies, we hypothesized that the biochemical alteration of tau thatinduced cognitive impairment would involve early pathological changes inthese specific molecular states. However, we did not observe anysubstantial correlations (i.e., r² >0.5) between biochemical expressionand retention of spatial reference memory (Table 2). Further analysis ofa panel of 12 antibodies failed to reveal close relationships betweenexpression of specific 55 kDa (full-length) tau isoforms and memoryfunction.

TABLE 2 Examples of epitopes that did not indicate a correlation betweenmemory retention and 55 kDa tau expression Antibody S1(extra-/intra-cellular) S2 (membrane-associated) MC-1 r² = 0.002 r² =0.006 CP-13 r² = 0.006 r² = 0.001 PHF-1 r² = 0.190 r² = 0.130 pThr²³¹ r²= 0.100 r² = 0.050 AT180 r² = 0.002 r² = 0.001

We turned our attention to other possible biochemical changes that mayunderlie tau-induced memory dysfunction. We considered, for example,changes in phosphorylation, conformation, solubility, oligomerization,and cleavage that may represent changes in tau biochemistry that inducecognitive impairment. Our rTg4510 mice lacked tau fragments reported byothers, including the tau fragment cleaved by multiple caspases at D421(Gamblin et al., PNAS, 2003, 100(17):10032-10037), the tau fragmentcleaved by caspase-6 at D402 (Guo et al., Am J Path, 2004), the 17-kDacalpain-cleaved tau fragment that is involved in Aβ-dependent death ofcultured neurons (Park & Ferreira, J Neurosci, 2005), and thethrombin-cleaved tau fragments found in tangles (Arai et al., J BlotChem, 2005).

In the course of performing these correlation experiments, weconsistently observed the presence of TCP35 in the brains of rTg4510mice, particularly in the hippocampus and cortex. The levels of TCP35were lower in cognitively intact age-matched rTg21221 mice expressingwild-type tau at levels equivalent to tau_(P301L) in rTg4510, arguingagainst the possibility that TCP35 was an artifact caused by proteindegradation while protein is being extracted from rTg4510 brain orduring gel electrophoresis. When we examined the relative expression ofTCP35 in relation to measures of behavior, we observed highlysignificant inverse relationships (FIG. 1).

Since memory deficits in rTg4510 mice develop in an age-dependentfashion, we also examined the expression of TCP35 at different ages andfound that the expression of TCP35 parallels the development of impairedmemory (FIG. 2). Taken together, the data indicate that TCP35 correlateswith cognitive impairment prior to the onset of neurodegeneration inrTg4510 mice.

Next, we characterized TCP35. On the basis of the immunospecificity ofTCP35 on western blots, we deduced that TCP35 is an N-terminal fragmentof tau (FIG. 3). Caspases typically cleave after aspartate residues,providing the opportunity to examine the molecular weights of a seriesof recombinant proteins mimicking tau, cleaved at the four aspartateresidues located in the region in tau that, when cleaved, would generatea fragment approximating 35 kDa. One truncation mutant, tauΔC314, mostclosely approximated TCP35 in molecular weight (FIG. 4).

We next assayed the ability of each of the eight different caspasesexpressed in the brain to cleave tau purified from brain extracts fromtau-expressing transgenic mice or synthesized using recombinant methods.Only one of the caspases, caspase-2, generated a 35 kDa tau fragment(FIG. 5), presumably recognizing the non-canonical cleavage sequenceKPVD₃₁₄ (SEQ ID NO:24), albeit cleaving with significantly lessefficiency than at D421. In most cells caspase-2 localizes to thenucleus, but in neurons caspase-2 is cytosolic, where it would haveaccess to tau, a necessary requirement for implicating this protease.

We then prepared tau mutants D314E and D314G that resisted caspase-2cleavage (FIG. 6), further supporting our hypothesis that TCP35 isgenerated when tau is cleaved by caspase-2 at D314.

Next, we generated H1485 polyclonal antibodies specifically recognizingthe C-terminal neo-epitope of tauΔ314 to probe brain extracts fromimpaired rTg4510 mice. In rTg4510 (Tau-positive) but not TgNeg mice, wefound a 35 kDa H1485-immunoreactive band that co-migrates with TCP35 inbrain proteins that were immunoprecipitated with Tau-13 mAbs (FIG. 7).The data provide further support for our hypothesis that caspase-2cleavage at tau D314 generates TCP35.

In some embodiments, TCP35 can include a polypeptide having the aminoacid sequence depicted in SEQ ID NO:16. In other embodiments, TCP35 caninclude a polypeptide having the amino acid sequence depicted in SEQ IDNO:17. The H1485 polyclonal antibody also recognizes TCP40 cleavageproducts (SEQ ID NO:14 and SEQ ID NO:15) and a TCP30 tau cleavageproduct (SEQ ID NO:18) (FIG. 13).

Tau cleavage product levels increase in patients with mild cognitiveimpairment and Alzheimer's disease. The protein levels of TCP35, TCP40,and TCP30 were quantified, respectively (FIG. 14). The total proteinlevels of TCP35, TCP40 and TCP30 (TCP) were also quantified.

Images of rat hippocampal neurons coexpressing Dsred and GFP-taggedwild-type tau or tau P301L mutant are shown in FIG. 15. Neurons weretreated with caspase-2 inhibitor Ac-VDVAD-CHO (SEQ ID NO:36) orcaspase-3 inhibitor Ac-DEVD-CHO (SEQ ID NO:37) for 1 day. FIG. 16 showsthe quantification of total spines and GFP-tau-containing spines inneurons coexpressing DsRed and GFP-tau. As the concentration ofcaspase-2 inhibitor increases, the number of spines with tau decreases.

The observations that (1) TCP35 appears at 4.5 months, when cognitiondeclines, (2) TCP35 exhibits a statistically significant correlationwith memory impairment, (3) TCP35 co-migrates with both tauΔ314 and atau fragment generated following incubation with caspase-2, and (4)tauΔ4C314 is present in rTg4510 but not TgNeg brain extracts suggestthat TCP35 is tau* and that caspase-2 cleaves tau at D314 to generateTCP35. These data, however, are correlative rather than causative. Thus,we next determined whether TCP35 causes neuronal dysfunction. We beganby assaying its effects on dendritic spines, which are loci of synapticplasticity underlying learning and memory.

Healthy neurons maintain a spatial gradient of tau, whose concentrationis greater in axons than in somatodendritic compartments. Inneurological disorders such as Alzheimer's disease, the gradient becomesinverted. One consequence of this inversion is aberrant accumulation oftau within intact dendritic spines, where it can disrupt synapticfunction by impairing glutamate receptor trafficking and/or synapticanchoring. We tracked chimeric tau molecules tagged at the N-terminuswith GFP and found that the P301L mutation andpseudo-hyperphosphorylation of tau both enhance its mislocalization tospines, with hyperphosphorylation appearing downstream of the mutationsince pseudo-hyperphosphorylation is sufficient to misdirect wild-typetau and blocking phosphorylation prevents the mislocalization oftau_(P301L). The abnormal presence of tau in spines corresponded to areduction in the clustering of both AMPA and NMDA receptors in spines,resulting in smaller amplitudes and frequencies of miniature excitatorypost-synaptic currents (mEPSCs).

In these experiments, we could not assess the role of TCP35 in mediatingtau mislocalization and subsequent synaptic impairment because theGFP-tagged tau variants did not distinguish between full-length andtruncated tau. In our initial experiments to determine whether TCP35 hasthe potential to disrupt neuronal function, we expressed GFP-taggedtauΔ314 in rat primary neuronal cultures and found that, liketau_(P301L) and pseudo-hyperphosphorylated tau, tauΔC314 alsomislocalizes to dendritic spines (FIG. 8). We noted that wild-type taudid not mislocalize, although caspase-2 cleaves wild-type recombinanttau in vitro. Caspase-2 cleavage of wild-type in neurons may involvephosphorylation.

The family of mammalian caspases shows close homology to the C. elegansapoptotic protein CED-3, and caspase-2 is the most conserved member ofthis family. Mammalian caspases possess many different functions beyondmediating apoptosis. Indeed, their non-death activities may beevolutionarily older than their lethal functions. For example, inaddition to its involvement in specific types of apoptosis, caspase-2also functions in DNA repair, cell cycle regulation and tumorsuppression. These cellular pathways may be involved in Alzheimer's andother neurodegenerative diseases.

To determine whether cleavage of tau by caspase-2 mediates taumislocalization in cultured neurons, one can examine tau trafficking inneurons derived from caspase-2 null mice, which are commerciallyavailable from vendors such as, for example, can be obtained from TheJackson Laboratory (Bar Harbor, Me.). One can determine whether theabsence of caspase-2 prevents the mislocalization ofpseudo-hyperphosphorylated tau and taup_(3o1L). If so, caspase-2 maymediate tau mislocalization.

TauΔC314 mislocalizes to spines (FIG. 8). Moreover, tau phosphorylationat 14 proline-directed serine and threonine sites (SP/TP) potentiatestau mislocalization. Conversely, preventing phosphorylation at the SP/TPsites blocks tau_(P301L) mislocalization. To determine the order of thecleavage and phosphorylation events, one can prepare a series ofGFP-tagged tauΔC314 and tau_(D314E) variants in which one can change theSP/TP residues to, for example, either (1) non-polar alanine residues toprevent phosphorylation, termed AP for alanine-proline, or (2)negatively charged glutamate residues to mimic phosphorylation, termedEP for glutamate-proline. Particularly informative data can be obtainedfrom experiments involving tauΔC314 in the AP tau variant andtau_(D314E) in the EP tau variant. For example, when cleavage followedby SP/TP phosphorylation results in tau mislocalization, then tauΔC314in the AP tau variant may not mislocalize to spines. When cleavage isdownstream of SP/TP phosphorylation, then mislocalization of tauΔC314may occur in both the AP and the EP tau variants. A practicalimplication of cleavage occurring downstream of hyperphosphorylation maybe the possibility of targeting caspase-2 rather than tau kinases. Sucha strategy may produce a lower chance of undesirable—even dangerous—sideeffects, given how few deficits occur in caspase-2 null mice.

Using the neurons generated to address the previous two questions, onecan examine the pattern of glutamate receptors (NR1, GluR1, GluR2/3) byimmunolocalization and measure the amplitude and frequency of mEPSCs bypatch clamping. These data can establish the extent to which thecleavage of tau by caspase-2 in neurons affects synaptic function.

The presence of tau in dendritic spines can mediate brain dysfunction inAβ and tau mouse models. However, contrasting mechanisms of action havebeen proposed: a) in one model it is the ability of tau in spines tostabilize NMDA receptor activity that mediates Aβ-induced memorydysfunction (Ittner et al., Cell, 2010); b) in another model it is theability of tau in spines to decrease NMDA and AMPA receptor activationthat mediates tau-induced memory dysfunction (Hoover et al., Neuron,2010). In neither model, however, has the specific form of tau thatmediates memory loss been identified in vivo. Thus, our identificationof TCP35 represents an advance regardless of the mechanism oftau-induced brain dysfunction.

One can assess the role of TCP35 by genetically ablating caspase-2 inrTg4510 mice, which should substantially limit—perhaps eveneliminate—the presence of TCP35. TCP35 levels can be monitored using,for example, Tau-13 mAb and H1485 neo-epitope antibodies. rTg4510 (“r”for regulatable) may be generated by mating activator (129S6-TgtTA) andresponder (FVB-Tg4510) mice. Caspase-2 null mice are in a B6 backgroundstrain. To obtain the desired mice, one can breed hemizygous caspase-2activator and responder mice in this exemplary cross:

129S6B6F1-TgtTA^(caspase-2Φ/+)×FVBB6F1-Tg4510^(caspase-2 Φ/+)

Only 1:16 offspring will be rTg4510^(caspase-2 Φ/Φ)orrTg4510^(caspase-2 +/+)mice.

Since caspase-2 null mice have intact memory function, one can usecaspase-2 null mice to determine whether caspase-2 is required forrTg4510 mice to develop memory deficits by comparing memory function inrTg4510 caspase-2 null (rTg4510 ^(caspase-2 Φ/Φ)) mice and rTg4510^(caspase-2+/+)littermates.

One also can perform longitudinal behavioral testing in these crosses toassess behavior both before and after the onset of deficits, in order totest the null hypothesis that there is no significant age by caspase-2interaction. One can test memory using a water maze. However, becausethe water maze can be difficult to interpret in longitudinal tests, onecan supplement the water maze with, for example, one or more of threeoperant tests: 1) fixed consecutive number test of cortical function; 2)delayed non-matching to sample test of hippocampal function; and 3)active avoidance test of hippocampal function.

Finally, one can examine human frontal cortex specimens for the presenceof TCP35. Suitable specimens include specimens obtained from, forexample, the Memory and Aging Project (MAP) from elderlycommunity-dwelling volunteers with no cognitive impairment, mildcognitive impairment, Alzheimer's disease, or frontotemporal dementia.An antibody such as, for example, H1485 neo-epitope antibodies totauΔC314 may be used to measure TCP35 in brain tissue from samplesobtained from subjects (e.g., MAP specimens). FIG. 12 shows theidentification of TCP35 in human cerebrospinal fluid obtained fromsubjects diagnosed with tauopathy.

In the preceding description, particular embodiments may be described inisolation for clarity. Unless otherwise expressly specified that thefeatures of a particular embodiment are incompatible with the featuresof another embodiment, certain embodiments can include a combination ofcompatible features described herein in connection with one or moreembodiments.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES

The rTg4510 Mouse Model

To study the etiology of dementia in tauopathy, we created a transgenicmouse model expressing the P301L mutation in human tau (tau_(P301L))linked to a dominantly inherited tauopathy. In an age dependent mannerthese mice develop: i) neurofibrillary pathology; ii) forebrain specificneurodegeneration; and iii) memory impairment and/or dementia.Pathological biochemical changes in tau are detected from 2.5 months ofage. The onset of memory deficits is first observed at 2.5 months and issignificant at 4 months. Mature neurofibrillary tangles appear at 4months. Significant neuronal loss is estimated by stereology to occur at5.5 months and is most striking in the hippocampus CA1 subfield. For acomprehensive description see Ramsden et al., J. Neurosci., 2005,25:10637-10647.

One feature of rTg4510, compared to many other tau transgenic mice, isthe preservation of motor function, achieved by engineering thetransgene to avoid expression in the brainstem, which permits the cleaninterpretation of cognitive tests uncomplicated by motor or sensoryabnormalities. Another feature of rTg4510 is that the first neurologicaldeficits appear at a few months of age, compared with one or two yearsof age in many other models. The early onset of tauopathy in rTg4510,achieved by overexpressing the human tau transgene, allows one toperform our studies relatively quickly.

Example 1 Animals

Briefly, rTg(tauP301L)4510 and rTg(tauWT)21221 mice were generated usinga system of responder and activator transgenes to achieve regulatableexpression (Hoover et al., Neuron, 2010). Mice expressing the activatortransgenes were derived following a generous gift from Dr. E. Kandel(Columbia University, New York, N.Y.) (Mayford et al., Science, 1996)and successively backcrossed a minimum of five times onto a 129S6background strain. Responder mice were maintained in the FVB/N strain.Mice were screened by PCR using the primer pairs5′-GATTAACAGCGCATTAGAGCTG-3′ (SEQ ID NO:6) and5′-GCATATGATCAATTCAAGGCCGATAAG-3′ (SEQ ID NO:7) for activator transgenesand 5′-TGAACCAGGATGGCTGAGCC-3′ (SEQ ID NO:8) and5′-TTGTCATCGCTTCCAGTCCCCG-3′ (SEQ ID NO:9) for responder transgenes.Doxycycline 200 ppm in chow was administered to mice ad libitum tosuppress transgene expression. The caspase-2 null (caspase-2ΦΦ) micewere purchased from the Jackson Laboratory and crossed with theactivator (129S6-TgtTA) and responder (FVB-Tg4510) mice separately. Theoffspring 129S6B6F1-TgtTA^(caspase-2Φ/−)andFVBB6F1-Tg4510^(caspase-2Φ/+)mice were further crossed to obtainrTg4510^(caspase-2Φ/Φ)and rTg4510^(caspase-2+/+)littermates. Allexperiments with animals were conducted in full accordance with theAmerican Association for the Accreditation of Laboratory Animal Care andInstitutional Animal Care and Use Committee at the University ofMinnesota.

Behavioral Analysis

Spatial reference memory was measured using the Morris water mazetailored to more rapid learning in the 129FVBF1 background strain(Westerman et al., J Neurosci, 2002). Mice were handled 60 seconds perday for 10 days during the 2 weeks before the initiation of testing.Prehandling was designed to condition the mice to manipulations thatwould be experienced during introduction and removal from the testingpool and included a 20 seconds exposure to water at a depth of 1 cm.Mice then received visible platform training for 3 days (six trials perday) and hidden platform training for 6 days (four trials per day). Thespatial cues and hidden platform location were changed at each agetested. Four probe trials of 30 seconds were performed 20 hours aftereither 8 hours, 12 hours, 16 hours, or 24 hours hidden training trials.The mean platform score and platform crossing index were calculated. Alltrials were monitored using a computerized tracking system (NoldusEthoVision 3.0; Noldus Information Technology, Wageningen, TheNetherlands), and performance measures were extracted using Wintrack(Wolfer et al., Physiol Behav, 2001).

Protein Extraction

Mouse brain tissue for biochemical studies was rapidly dissected andquickly frozen for storage at −80° C. To generate forebrain lysates,olfactory bulbs, corticolimbic and subcortical brain stem structures,and the cerebellum were all removed. Frozen hemi-forebrains were thawedand mixed with 1 ml of solution containing 50 mM Tris-HCl (pH 7.4), 150mM NaCl, 1% Triton X-100, 3% SDS, 1% deoxycholate, phosphate inhibitorcocktail, 0.2 mM 1,10-phenanthroline, 1 mM phenylmethylsulfonylfluoride, and protease inhibitor cocktail (Sigma-Aldrich, St. Louis,Mo.). Soluble proteins were collected from mechanically homogenizedlysates (1 ml syringe, 20 gauge needle [10 repeats]) followingcentrifugation for 90 minutes at 13,000 rpm. Protein concentrations weremeasured by using Pierce BCA protein assay kit (Thermo FisherScientific, Waltham, Mass.) following manufacturer's instruction.

DNA Constructs

All tau constructs used for transfecting neuronal cultures were taggedwith GFP on the N terminus and expressed in the pRK5 vector and drivenby a cytomegalovirus (CMV) promoter (Clontech Laboratories, Inc.,Mountain View, Calif.) as described in Hoover et al., Neuron 2010. TheGFP and DsRed constructs (Clontech Laboratories, Inc., Mountain View,Calif.) were also expressed in the pRK5 vector and driven by a CMVpromoter. The full-length tau construct encoded human four-repeat taulacking the N-terminal region (4R0N) and contained exons 1, 4 and 5, 7,and 9-13, intron 13, and exon 14. The GFP-tagged ΔC314 construct wasgenerated from the full-length tau sequence by mutating Leu315 residueto a stop codon with a QuikChange site-directed mutagenesis kit(Stratagene, Agilent Technologies, Inc., Santa Clara, Calif.). Tosynthesize recombinant tau proteins, full-length tau was subcloned frompRK5 vector into pcDNA3 vector (Invitrogen, Life Technologies Corp.,Carlsbad, Calif.) by using BamHI and XhoI restriction sites. A series oftruncation mutants including ΔC348, ΔC314, ΔC295 and ΔC283 weregenerated from the full-length tau construct by mutating Arg349, Leu315,Asn296 and Leu284 to stop codons, respectively. The tau D314E mutant wasgenerated from the full-length tau construct by mutating Asp314 to Glu.Using full-length tau as a template, two tau constructs termed AP or EPwere generated by mutating all 14 S/P or T/P amino acid residues (T111,T153, T175, T181, S199, S202, T205, T212, T217, T231, S235, S396, S404,and S422 to Ala (AP) or Glu (EP). All coding sequence authenticity wasconfirmed by DNA sequencing performed by BioMedical Genomics Center,University of Minnesota.

Caspase Cleavage Assay

Tau proteins (200 ng immunoprecipitates from rTg[tauP301L]4510 orrTg[tauWT]21221 brain lysates, or 1 μl synthetic proteins generated withTNT T7 Quick Coupled Transcription/Translation System [Promega Corp.,Madison, Wis.]) were diluted in assay buffer (20 mM HEPES pH 7.2, 10 mMDTT, 1 mM EDTA, 0.1% CHAPS) to a final volume of 50 μl. One unit ofrecombinant caspase was added to the reactions in the presence orabsence of 50 μM z-VAD-fmk (Calbiochem, EMD Biochemicals, San Diego,Calif.). The reactions were incubated at 37° C. for 1.5 hours andstopped by adding Laemmli sample buffer (62.5 mM Tris-HC1 [pH 6.8], 25%glycerol, 2% SDS, and 0.01% bromophenol blue). The reactions were thensubjected to western blotting and revealed with Tau-13 antibody (CovanceInc., Princeton, N.J.).

Western Blotting and Densitometric Analysis

Five micrograms of rTg(tauP301L)4510 mouse brain lysates wereelectrophoresed on 10% Tris-HCl gels (Bio-Rad Laboratories, Inc.,Hercules, Calif.), then transferred onto 0.45 μm polyvinylidenedifluoride membranes (Millipore Corp., Billerica, Mass.). Membranes wereblocked in 5% BSA and probed with primary antibody Tau-13 (Covance Inc.,Princeton, N.J.), and visualized using enhanced chemiluminescencereagents (Thermo Fisher Scientific, Waltham, Mass.) followed by exposureonto hyperfilm (Kodak). βIII-Tubulin was also probed as loadingcontrols. Densitometric analysis of protein signal was performed usingthe ImageJ software (NIH).

Tau H1485 Antibody Production

Rabbit polyclonal H1485 antibody was generated against a cleavagepeptide corresponding to the C terminus of tau truncated at Asp314 byusing custom antibody service (New England Peptide LLC, Gardner, Mass.).Specifically, the peptide Ac-CIVYKPVD-OH (SEQ ID NO:10), whichcorresponds to tau residues 308-314 with a Cys added to the N terminus,was synthesized. This peptide was coupled through the Cys to keyholelimpet hemocyanin. Rabbits were immunized with the cleavage peptidethree times over a period of four weeks. Seven and twelve days after thefinal immunization, the rabbits were bled and their sera were collected,respectively. The sera were combined and incubated with an affinitycolumn using a spanning peptide (Ac-CIVYKPVDLSKVT-amide, SEQ ID NO:11)which corresponds to tau residues 308-319. The flow-through from thiscolumn was then incubated with an affinity column using the cleavagepeptide. The cleavage-site specific antibody H1485 was then eluted offthe column and stored at −20° C.

Immunoprecipitation Assay

Fifty micrograms of rTg[tauP301L]4510 brain lysates were diluted in 1 mllysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.2 mM1,10-phenanthroline, 1 mM phenylmethylsulfonyl fluoride, and proteaseinhibitor cocktail) and incubated at 4° C. overnight under agitation inthe presence of 5 μg Tau-13 antibody. Twenty microliters of Protein GSepharose beads (GE Healthcare, UK) were added to the samples. Thesamples were further incubated for 2 hours and then centrifuged. Thesupernatant was discarded and the beads were washed in lysis bufferthree times. Finally, the proteins were eluted off by adding 40 μlLaemmli sample buffer to the beads. The immunoprecipitated proteins wereanalyzed with western blotting.

Primary Neuronal Culture and Transfection

Based on methods described in Hoover et al., Neuron 2010, dissociatedrat hippocampal primary neuron cultures were prepared. Briefly, a 25 mmglass coverslip (thickness, 0.08 mm) was glued over a 22 mm hole in thebottom of a 35 mm tissue culture dish using silicone sealant.Dissociated neuronal cultures from rat hippocampi at P1 were prepared.Neurons were plated onto prepared 35 mm tissue culture dishes at adensity of 1×10⁶ cells per dish. The age of cultured neurons was countedfrom the day of plating (1 DIV). Neurons at 7 DIV were transfected usinga standard calcium phosphate precipitation method and allowed to grow tomaturity (>3 weeks) to be imaged. Neurons were transfected withequivalent concentrations of plasmids encoding full-length tau or tauΔC314 mutant. The plasmid encoding DsRed was cotransfected with tauconstructs to visualize dendritic spines.

Image Analysis of Living Neurons

Coverslips of neurons cotransfected with various GFP-tagged tauconstructs and DsRed were photographed on an inverted Nikonepifluorescent microscope with a 60×oil lens and a computerized focusmotor at 21-35 DIV. All digital images were photographed and processedwith MetaMorph Imaging System (Universal Imaging Corporation, WestChester, Pa.). All images were taken as stacks (15 planes at 0.5 micronincrements) and processed by deconvolution analyses using the MetaMorphsoftware with the nearest planes and averaged into one single image. Adendritic protrusion with an expanded head that was 50% wider than itsneck was defined as a spine.

Electrophysiological Analysis

Miniature excitatory postsynaptic potentials (mEPSCs) were recorded at−55 mV in artificial cerebrospinal fluid (aC SF) containing 100 μM AP7(NMDAR antagonist, Tocris Bioscience, UK), 1 μM TTX (sodium channelblocker, Tocris Bioscience, UK) and 50 μM bicuculline (GABA receptorantagonist, Sigma-Aldrich, St. Louis, Mo.). Electrophysiologyexperiments were conducted using AXON DIGIDATA 1440A data acquisitionsystem (Molecular Devices, LLC, Sunnyvale, Calif.), AXOPATCH 200Bamplifier (Molecular Devices, LLC, Sunnyvale, Calif.), pCLAMP10 software(Molecular Devices, LLC, Sunnyvale, Calif.). Data was analyzed usingAxoGraph X software (Berkeley, Calif.).

Screening Assay

Unless otherwise stated, chemicals were purchased from Sigma-Aldrich(St. Louis, Mo.). Caspase-2 was made in-house; reference Caspase-2inhibitor Z-VAD-FMK was purchased from BD Pharmingen (San Diego,Calif.). The fluorescently labeled substrate for Caspase-2,FITC-AHX-GSVQIVYKPVDLSKVTS-COOH, was custom synthesized by CeltekPeptides (Franklin, Tenn.). Assay buffer contained 20 mM HEPES, pH 7.2,1 mM EDTA, 0.1% CHAPS, and 10 mM DTT.

In black, Greiner 384-well plates, 80 μL of assay buffer was added toeach well; wells that were not screened for compounds contained onlythis buffer. For reagent wells, 4 μL of compound (or 4 μL of buffer as acontrol) and 15 μL of Caspase-2 (30 U/μL) were added to the buffer.After a 10-minute incubation at room temperature, 1 μL of 0.5 mMsubstrate in 50% DMSO was added to each reagent well (for a finalsubstrate concentration of 5 μM/well); plates were then incubated at 37°C. After 1.5 hours of incubation, 10 μL of stop solution (8.0 pH buffercontaining 0.1% coating-3 reagent (Caliper Life Sciences Inc.,Hopkinton, Mass.)) was added to each well.

Plates were screened in a LabChip 3000 (Caliper Life Sciences Inc.,Hopkinton, Mass.) with temperature and relative humidity set to 20° C.and 50%. A 12-sipper chip sampled contents from each well and measuredthe fluorescent intensity of the substrate and product peaks. Foroptimal electrophoretic separation of substrate and product peaks, aseparation buffer consisting of ProfilerPro separation buffer and 0.1%coating-3 reagent (Caliper Life Sciences Inc., Hopkinton, Mass.) was runthrough the LC3000, and a pressure of −1.0 psi and a voltage of -1700ΔV(−1200V/−2900V) were applied during the screening process. Data wereanalyzed using Caliper's HTS Well Analyzer software.

Example 2 Protein Extraction

Mouse brain tissue for biochemical studies was rapidly dissected andquickly frozen for storage at −80° C. To generate forebrain lysates,olfactory bulbs, corticolimbic and subcortical brain stem structures,and the cerebellum were all removed. Frozen hemi-forebrains were thawedand mixed with 1 ml of solution containing 50 mM Tris-HCl (pH 7.4), 150mM NaCl, 1% Triton X-100, 3% SDS, 1% deoxycholate, phosphate inhibitorcocktail, 0.2 mM 1,10-phenanthroline, 1 mM phenylmethylsulfonylfluoride, and protease inhibitor cocktail (Sigma). Soluble proteins werecollected from mechanically homogenized lysates (1 ml syringe, 20 gaugeneedle [10 repeats]) following centrifugation for 90 min at 13,000 rpm.Protein concentrations were measured by using Pierce BCA protein assaykit (Thermo Fisher Scientific, Waltham, Mass.) following manufacturer'sinstruction.

A cohort of 85 human tissue samples was obtained from the Memory andAging Project, Rush University. These samples represent elderly peoplewith no cognitive impairment (NCI), mild cognitive impairment (MCI) andAlzheimer's disease (AD). The prefrontal cortex of these samples washomogenized in solution containing 50 mM Tris-HCl (pH 7.6), 0.01% NP-40,150 mM NaCl, 2mM EDTA, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, andprotease inhibitor cocktail (Sigma-Aldrich, St. Louis, Mo.) andcentrifuged for 90 min at 3,000 rpm. The pellet was further extracted insolution containing 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% TritonX-100 and centrifuged for 90 min at 13,000 rpm. Protein concentrationsof the supernatant were measured by using Pierce BCA protein assay kit(Thermo Fisher Scientific, Waltham, Mass.) following manufacturer'sinstruction. Results are shown in FIG. 14.

Immunoprecipitation Assay

Fifty micrograms of rTg[tauP301L]4510 brain lysates were diluted in 1 mllysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.2 mM1,10-phenanthroline, 1 mM phenylmethylsulfonyl fluoride, and proteaseinhibitor cocktail) and incubated at 4° C. overnight under agitation inthe presence of 5 μg Tau-13 antibody (Covance Inc., Princeton, N.J.).Three hundred micrograms of human tissue lysates were diluted in 1 mllysis buffer and incubated at 4° C. overnight under agitation in thepresence of 30 μg Tau-13 antibody (Covance Inc., Princeton, N.J.).Twenty microliters of Protein G Sepharose beads (GE Healthcare, UK) wereadded to the samples. The samples were further incubated for 2 h andthen centrifuged. The supernatant was discarded and the beads werewashed in lysis buffer three times. Finally, the proteins were elutedoff by adding 40 pi Laemmli sample buffer to the beads. Theimmunoprecipitated proteins were analyzed with western blotting by usingH1485 antibody.

Western Blotting and Densitometric Analysis

Five micrograms of rTg(tauP301L)4510 mouse brain lysates wereelectrophoresed on 10% Tris-HCl gels (Bio-Rad Laboratories, Inc.,Hercules, Calif.), then transferred onto 0.45 μm polyvinylidenedifluoride membranes (Millipore Corp., Billerica, Mass.). Membranes wereblocked in 5% BSA and probed with primary antibody Tau-13 (Covance Inc.,Princeton, N.J.), and visualized using enhanced chemiluminescencereagents (Thermo Fisher Scientific, Waltham, Mass.) followed by exposureonto hyperfilm (Kodak). Human tissue lysates were probed with H1485antibody, 8E6/C11 antibody (Millipore), 1E1/A6 antibody (Millipore), andTau-13 antibody(Covance Inc., Princeton, N.J.). Densitometric analysisof protein signal was performed using the ImageJ software (NIH). Resultsare shown in FIG. 13.

Primary Neuronal Culture and Transfection

Based on methods described in Hoover et al., Neuron 2010, dissociatedrat hippocampal primary neuron cultures were prepared. Briefly, a 25 mmglass coverslip (thickness, 0.08 mm) was glued over a 22 mm hole in thebottom of a 35 mm tissue culture dish using silicone sealant.Dissociated neuronal cultures from rat hippocampi at P1 were prepared.Neurons were plated onto prepared 35 mm tissue culture dishes at adensity of 1×10⁶ cells per dish. The age of cultured neurons was countedfrom the day of plating (1 DIV). Neurons at 7 DIV were transfected usinga standard calcium phosphate precipitation method and allowed to grow tomaturity (>3 weeks) to be imaged. Neurons were transfected withequivalent concentrations of constructs encoding various tau proteins.The plasmid encoding DsRed was cotransfected with tau constructs tovisualize dendritic spines. Recombinant tau protein or TCP35 protein (10μM) was directly added to culture medium 1 hour before imaging tostimulate neuronal cultures. Caspase-2-selective inhibitor Ac-VDVAD-CHO(SEQ ID NO:36; Sigma-Aldrich, St. Louis, Mo.) or caspase-3-selectiveinhibitor Ac-DEVD-CHO (SEQ ID NO:37; Promega Corp., Madison, Wis.) wasdirectly added to culture medium 2 days before imaging to blockcaspase-2 or caspase-3 activity.

Image Analysis of Living Neurons

Coverslips of neurons cotransfected with various GFP-tagged tauconstructs and DsRed were photographed on an inverted Nikonepifluorescent microscope with a 60×oil lens and a computerized focusmotor at 21-35 DIV. All digital images were photographed and processedwith MetaMorph Imaging System (Universal Imaging Corporation, WestChester, Pa.). All images were taken as stacks (15 planes at 0.5 micronincrements) and processed by deconvolution analyses using the MetaMorphsoftware with the nearest planes and averaged into one single image. Adendritic protrusion with an expanded head that was 50% wider than itsneck was defined as a spine. Results are shown in FIG. 15 and FIG. 16.

Expression and Purification of Recombinant Protein in E. coli

Recombinant tau and TCP35 proteins were expressed by using pET system(Novagen, EMD Millipore, Billerica, Mass.) following manufacturer'sinstruction. Briefly, the coding sequences of tau and TCP35 were clonedinto pET28a vector, respectively. The expression constructs weretransformed in BL21(DE3) E. coli. The expression of recombinant proteinswas induced by 1 mM isopropyl β-D-1-thiogalactopyranoside at 37° C. for3 hours. The recombinant proteins were purified from E. coli celllysates by using immobilized metal affinity chromatography (ThermoFisher Scientific, Waltham, Mass.) following manufacturer's instruction.

Example 3

The pentapeptides shown in Table 1 were synthesized using a solidsupport and accompanied by microwave irradiation to heat the reactionmixture during coupling and N(α)-deprotection.

Solid Phase Peptide aldehyde Synthesis

The pentapeptides were synthesized using 0.05 mmol (250 mg) scale ofH-asp(OtBu)-H preloaded aldehyde resin (AnaSpec. Inc., Fremont, Calif.)following the manufacturer's instructions. Briefly, the H-asp(OtBu)-Hpreloaded resin allows the synthesis of peptides containing C-terminalaspartinal. The peptide aldehyde synthesis is carried out using standardinstrument protocols. Cleavage from the resin and side-chaindeprotection is performed in two stages. It enables side-products fromside-chain deprotection to be removed by washing before the product isreleased into aqueous solution.

The resin was added to a 20 mL fritted glass tube and was swollen for2-16 hours in DCM (dichloromethane; Sigma-Aldrich, St. Louis, Mo.). TheDCM was then removed from resin and it was washed 3 times with DMF(N,N′-dimethylformamide from Sigma-Aldrich, St. Louis, Mo.) using oneminute mix times.

Amino acids were coupled using FmocAA (9-N-fluorenylmethyloxycarbonylamino acids from Sigma-Aldrich, St. Louis Mo.) (3 equivalents), HOBt(hydroxybenzotriazole from Sigma-Aldrich, St. Louis, Mo.) 3.1equivalents, 0.155 mmol, 21 mg), DIC (diisopropopylcarbodiimide fromSigma-Aldrich, St. Louis, Mo.) (3.2 equivalents, 0.16 mmol, 25 μL) weredissolved in the same vial in anhydrous DMF (2-3 mL) and then added tothe resin. The resin pre-stirred for 30 seconds, then heated to 65° C.for 15 minutes in a 55W microwave oven. The resin was filtered andwashed three times with 3 mL aliquots of DMF using 1 minute mix time foreach. The ninhydrin test (AnaSpec. Inc., Fremont, Calif.) was performedto evaluate the completion of the coupling reaction according to themanufacturer's instructions.

To deprotect the N-terminal α-amine group, a 20% v/v solution ofpiperidine in DMF (0.6 mL/3 mL) was added to the resin. The resinpre-stirred for 30 seconds, then heated to 65° C. for 10 minutes in a55W microwave oven. The resin was filtered and washed three times with 3mL aliquots of DMF (1 minute each). The ninhydrin test was performedaccording to the manufacturer's instructions, the resin was rinsed threetimes with DCM (3 mL) and then dried under vacuum for 10 minutes.

If a terminal N-Ac-aa was not commercially available, following the lastdeprotection, a solution of aceticanhydride/N,N′-diisopropylethylamine/N,N′-dimethylformamide(Ac₂O/DIPEA/DMF, 3/1/96 (5 mL), all components from Sigma-Aldrich, St.Louis, Mo.) was added to the resin and the mixture was stirred for 15minutes at room temperature. The resin was then rinsed three times withDCM and dried under vacuum.

Deprotection of lateral chains was performed using 3 mL of TFA was addedin the fritted glass tube and the mixture was stirred bubbled withnitrogen for 30 minutes at room temperature. The resin was filtered andwashed with DCM (5 mL).

Finally a solution of CH₃CN/water (60/40; v/v) with 0.1%TFA was used forthe cleavage from the resin. 5 mL of the solution were added and themixture was stirred twice for 30 minutes at room temperature followed byfiltration and washing with two further aliquots of mixture CH₃CN/water(60/40) with 0.1% TFA. The filtrate was lyophilized to give the crudeproduct which was purified using a preparative HPLC (GEMINI 5 μm C18110A, 150×10 mm from Phenomenex Inc., Torrence, Calif.; gradient 5% to100% of acetonitrile in water +0.1% formic acid over 10 minutes) toafford the final pentapeptide aldehyde product.

Example 4 Tau Aptamer Production

Sequences for aptamer selection are prepared using standard peptidechemistry or resourced. The sequence for selection can be as short as a5-mer (e.g., SEQ ID NO:1) or as long as the 314-amino acid cleavageproduct (e.g., SEQ ID NO: 13), and are chosen based on empiricalresults. Selection of aptamers is performed using systematic evolutionof ligands by an exponential enrichment (SELEX) methods as previouslydescribed (Takemura, et al. Exp. Biol. Med. (Maywood) 2006, 231:204-214;Wongphatcharachai, et al. J. Clin. Microbiol. 2013, 51:46-54.).Counter-selection is performed using the full-length tau protein.

As used herein, the term “and/or” means one or all of the listedelements or a combination of any two or more of the listed elements; theterms “comprises” and variations thereof do not have a limiting meaningwhere these terms appear in the description and claims; unless otherwisespecified, “a,” “an,” “the,” and “at least one” are used interchangeablyand mean one or more than one; the recitations of numerical ranges byendpoints include all numbers subsumed within that range (e.g., 1 to 5includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.); and reference torelative terms such as, for example, increase, decrease, or delay refersto comparison with an appropriate control including, where appropriate,an individual or population of individuals not receiving the indicatedtreatment.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety. In theevent that any inconsistency exists between the disclosure of thepresent application and the disclosure(s) of any document incorporatedherein by reference, the disclosure of the present application shallgovern. The foregoing detailed description and examples have been givenfor clarity of understanding only. No unnecessary limitations are to beunderstood therefrom. The invention is not limited to the exact detailsshown and described, for variations obvious to one skilled in the artwill be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

Sequence Listing Free Text SEQ ID NO: 1 YKPVD SEQ ID NO: 2AHX-GSVQIVYKPDLSKVTS SEQ ID NO: 3 AHX-GSVQIVYKPVD SEQ ID NO: 4AHX-GSVQIVYK(acetyl)PVDLSKVTS SEQ ID NO: 5 AHX-GSVQIVYK(acetyl)PVD SEQID NO: 6 GATTAACAGC GCATTAGAGC TG SEQ ID NO: 7 GCATATGATC AATTCAAGGCCGATAAG SEQ ID NO: 8 TGAACCAGGA TGGCTGAGCC SEQ ID NO: 9 TTGTCATCGCTTCCAGTCCC CG SEQ ID NO: 10 CIVYKPVD SEQ ID NO: 11 CIVYKPVDLSKVT SEQ IDNO: 12 IVYKPVD SEQ ID NO: 13 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTDAGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEGTTAEEAGIGD TPSLEDEAAG HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPPGQKGQANATR IPAKTPPAPK TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPTREPKKVAVVR TPPKSPSSAK SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINKKLDLSNVQSK CGSKDNIKHV PGGGSVQIVY KPVD SEQ ID NO: 14 MAEPRQEFEVMEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG SETSDAKSTPTAEAEEAGIG DTPSLEDEAA GHVTQARMVS KSKDGTGSDD KKAKGADGKT KIATPRGAAPPGQKGQANAT RIPAKTPPAP KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR SRTPSLPTPPTREPKKVAVV RTPPKSPSSA KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ PGGGKVQIINKKLDLSNVQS KCGSKDNIKH VPGGGSVQIV YKPVD SEQ ID NO: 15 MAEPRQEFEVMEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG SETSDAKSTPTAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG HVTQARMVSKSKDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK TPPSSGEPPKSGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAK SRLQTAPVPMPDLKNVKSKI GSTENLKHQP GGGKVQIVYK PVD SEQ ID NO: 16 MAEPRQEFEV MEDHAGTYGLGDRKDQGGYT MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA AGHVTQARMV SKSKDGTGSDDKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPA PKTPPSSGEP PKSGDRSGYSSPGSPGTPGS RSRTPSLPTP PTREPKKVAV VRTPPKSPSS AKSRLQTAPV PMPDLKNVKSKIGSTENLKH QPGGGKVQII NKKLDLSNVQ SKCGSKDNIK HVPGGGSVQI VYKPVD SEQ ID NO:17 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPGSETSDAKSTP TAEAEEAGIG DTPSLEDEAA GHVTQARMVS KSKDGTGSDD KKAKGADGKTKIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPP KSGDRSGYSS PGSPGTPGSRSRTPSLPTPP TREPKKVAVV RTPPKSPSSA KSRLQTAPVP MPDLKNVKSK IGSTENLKHQPGGGKVQIVY KPVD SEQ ID NO: 18 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYTMHQDQEGDTD AGLKAEEAGI GDTPSLEDEA AGHVTQARMV SKSKDGTGSD DKKAKGADGKTKIATPRGAA PPGQKGQANA TRIPAKTPPA PKTPPSSGEP PKSGDRSGYS SPGSPGTPGSRSRTPSLPTP PTREPKKVAV VRTPPKSPSS AKSRLQTAPV PMPDLKNVKS KIGSTENLKHQPGGGKVQIV YKPVD

1-48. (canceled)
 49. A method of detecting a product ofcaspase-2-mediated cleavage of tau in a subject, the method comprising:contacting a biological sample comprising cerebral spinal fluid from thesubject; and detecting the polypeptide of SEQ ID NO:13 in the biologicalsample.
 50. The method of claim 49 wherein detecting SEQ ID NO:13comprises: contacting the sample with an antibody preparation thatspecifically binds to SEQ ID NO:13; allowing the antibody preparation tobind to the polypeptide of SEQ ID NO:13 in the biological sample;removing unbound antibody preparation; and detecting the antibodypreparation that has bound to SEQ ID NO:13.
 51. The method of claim 50wherein the antibody preparation comprises a polyclonal antibodypreparation.
 52. The method of claim 51 further comprising quantifyingSEQ ID NO:13 in the biological sample.
 53. The method of claim 49wherein the biological sample is a first biological sample and themethod further comprises detecting the product of caspase-2-mediatedcleavage of tau in a second sample obtained from the subject.
 54. Themethod of claim 53 wherein detecting the product of caspase-2-mediatedcleavage of tau in a second sample obtained from the subject comprises:contacting a second biological sample comprising cerebral spinal fluidfrom the subject; and detecting the polypeptide of SEQ ID NO:13 in thesecond biological sample.
 55. The method of claim 54 wherein detectingSEQ ID NO:13 in the second biological sample comprises: contacting thesecond biological sample with an antibody preparation that specificallybinds to SEQ ID NO:13; allowing the antibody preparation to bind to thepolypeptide of SEQ ID NO:13 in the second biological sample; removingunbound antibody preparation; and detecting antibody preparation thathas bound to SEQ ID NO:13 in the second biological sample.
 56. Themethod of claim 54 further comprising quantifying SEQ ID NO:13 in thesecond biological sample.
 57. The method of claim 54 wherein the firstbiological sample and the second biological sample are obtained from thesubject at different times.